The androgen receptor (AR) is the single best molecular target for prostate cancer (PCa). Novel anti-androgens will likely be useful in treating both primary and recurrent PCa. Such anti-androgens need not be competitive antagonists, and could conceivably block downstream events in AR signaling. We have used cellular screens to identify two novel, potent anti-androgens: one is an FDA-approved drug, and the other a natural product. These compounds function as non-competitive antagonists, and synergize with classical anti-androgens such as hydroxy-flutamide or bicalutamide. Preliminary evidence suggests one compound is effective in vivo, especially in combination with BiC. The long term goals of this grant are to develop a better therapy for PCa. Aim 1: Efficacy studies in mice. We will treat animals for via IP injection, testing the candidate compound with bicalutamide alone or in combination, with castration as a positive control. We will measure effects on prostate weight and morphology, and will use RT-PCR to determine the degree to which it can down-regulate AR-dependent gene expression in the prostate gland. If successful, we will then establish an optimal ratio of the compound with bicalutamide to achieve maximal responses. These studies will underlie translation of this compound to the clinic for the treatment of hormone-refractory PCa in humans. Aim 2: Determine the mechanism of action of lead compounds. Both compounds identified have marked potency in cellular systems. Determination of their molecular mechanism of action will help elucidate biological mechanisms that control AR activity, and may lead to new therapeutic targets. We will use the extensive knowledge of steroid receptor biology to characterize at which step compounds inhibit AR activity. We will use FRET-based assays to determine effects on intramolecular and intermolecular conformational change. We will test for effects on AR nuclear localization via cell fractionation and western blot. Since the compounds specifically inhibit AR, but not the closely related glucocorticoid receptor, we will use domain transposition to test which regions of AR mediate its sensitivity to inhibition. This will facilitate identification of factors that interact with these regions, and which may be modulated by the compounds. We will measure effects of test compounds on gene regulation and promoter occupancy using quantitative RT-PCR and chromatin immunoprecipitation (ChIP) to determine whether they affect AR transcription globally, or only at specific subsets of promoters. These approaches will pinpoint the precise mechanisms of AR inhibition, will facilitate identification of intracellular targets, and will expand our knowledge of AR biology, facilitating new treatment strategies for PCa.